Neurofascin155 (NF155) accumulates in that lateral edges of the myelin sheath and tethers the sheath to the axon by binding the neuronal heterodimer of contactin/caspr. In early stages of multiple sclerosis (MS), NF155 clusters are lost resulting in compromised myelin-axon adhesion leading to demyelination and the development of devastating motor and cognitive deficits. Thus, maintaining NF155 cluster stability is critical for preserving neuronal function. Unfortunately, the mechanisms that regulate NF155 cluster stability are unknown. Based on preliminary data from my laboratory, I have identified 2 potential methods of NF155 anchoring that may play essential, yet temporally distinct, roles in maintaining NF155 clusters and myelin stability. My initial findings suggest that intramembrane interactions between NF155 and lipids regulate the position of NF155 in the myelin sheath during myelin formation. Perhaps surprisingly, the actin cytoskeletal network does not appear to play a role in NF155 domain stabilization during this stage of development. With age, however, NF155 domain stability appears to be dependent on the actin network. It is yet to be determined what role, if any, the NF155-lipid interactions play in the mature sheath. To confirm these findings, I will employ a modified version of an in situ extraction method to disrupt either lipid domains or the actin network. Upon confirming specific disruption, I will use a variety of detergents to test NF155 cluster stability. Based on published work from my laboratory, lipids, particularly the myelin glycosphingolipid sulfatide, are required for maintaining NF155 clusters. To elucidate how lipids regulate NF155 distribution, I will generate several fluorescently labeled NF155 constructs with and without mutations to amino acids required for palmitoylation, a post translation modification known to mediate protein-lipid interactions. Following transfection of these constructs, I will employ Fluorescent Recovery After Photobleaching to quantify the role that palmitoylaiton plays in the regulation of NF155 cluster maintenance. PUBLIC HEALTH RELEVANCE: Neurofascin155 clusters in the paranode of the myelin sheath and tethers the sheath to the axon. In early stages of multiple sclerosis, these paranodal clusters are lost facilitating demyelination and subsequent motor and cognitive deterioration. The studies outlined in this proposal are designed to identify mechanisms that regulate NF155 distribution as these appear to be targets of the disease process. Identification of these disease targets should facilitate the development of novel therapeutic strategies designed to ameliorate the effects of this devastating disease.